Anionic acid-labile surfactants and methods of use

ABSTRACT

Anionic acid-labile surfactants may generally comprise compounds represented by the formula: 
     
       
         
         
             
             
         
       
     
     wherein R 1  is independently selected from —(CH 2 ) 0-9 CH 3 , R 2  is selected from the group consisting of —H and —(CH 2 ) 0-5 CH 3 , Y is an anion, X is a cation, and n is an integer from 1 to 8. Methods of making and using the anionic acid-labile surfactants are also described. The anionic acid-labile surfactants may be used to facilitate the solubilization of proteins and other molecules in an aqueous environment.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.13/195,809, filed on Aug. 1, 2011, now U.S. Pat. No. ______, which is adivisional of U.S. application Ser. No. 12/454,742, filed on May 22,2009, now U.S. Pat. No. 8,013,179, which claims the benefit of U.S.Provisional Application No. 61/055,746, filed on May 23, 2008. Each ofthe foregoing applications is incorporated herein by reference in itsentirety.

BACKGROUND

The compounds and methods described herein generally relate to anionicacid-labile surfactants and methods of use.

Proteomics is the study of the structure and function of proteins andother molecules in biological systems. Some purification andidentification techniques used in proteomics require the proteins andother molecules to be solubilized in an aqueous environment. Mostproteins and other hydrophobic molecules or molecules with significanthydrophobic regions, however, are not readily soluble in an aqueousenvironment. A surfactant or detergent may generally be used tofacilitate the solubilization of proteins and other molecules in anaqueous environment.

Ionic cleavable surfactants have been successfully used to facilitatethe solubilization of proteins and other molecules in an aqueousenvironment. Ionic cleavable surfactants may generally comprise a polar(hydrophilic) group joined by a cleavable linker to a non-polar(hydrophobic) group. The ionic cleavable surfactant may be cleaved ordegraded by utilizing acidic conditions, basic conditions,photodegradation, thermal degradation, or chemical reduction. Thecleavage by-products may be separated from the proteins or othermolecules using standard isolation techniques. Conventional ioniccleavable surfactants, however, may generally comprise chemicalstructures that are complex to synthesize, require harsh conditions(e.g., pH 1-2) or long periods of time (up to several hours) to cleave,and/or generate cleavage by-products that interfere with purificationand identification techniques.

Accordingly, more efficient ionic cleavable surfactants and methods ofuse are desirable.

SUMMARY

According to certain embodiments, more efficient ionic cleavablesurfactants and methods of use are described.

In certain embodiments, anionic acid-labile surfactants may generallycomprise a compound of the formula:

wherein R₁ is independently selected from —(CH₂)₀₋₉CH₃, R₂ is selectedfrom the group consisting of —H and —(CH₂)₀₋₅CH₃, Y is an anion, X is acation, and n is an integer from 1 to 8.

In certain embodiments, methods of using the anionic acid-labilesurfactants may generally comprise adjusting a sample to pH 6-12; mixinga solvent with at least one of the anionic acid-labile surfactants;contacting the sample with the mixture; and cleaving the at least oneanionic acid-labile surfactant.

DESCRIPTION OF THE DRAWINGS

The various non-limiting embodiments of anionic acid-labile surfactantsand methods of use described herein may be better understood byconsidering the following description in conjunction with theaccompanying drawings.

FIG. 1 illustrates the degradation time of an embodiment of an anionicacid-labile surfactant and three conventional anionic acid-labilesurfactants.

FIG. 2 illustrates the protein score from in-solution digestion of BSAof an embodiment of an anionic acid-labile surfactant and threeconventional anionic acid-labile surfactants.

FIG. 3 illustrates the protein score from in-solution digestion ofovalbumin of an embodiment of an anionic acid-labile surfactant andthree conventional anionic acid-labile surfactants.

DESCRIPTION OF CERTAIN EMBODIMENTS A. Definitions

As generally used herein, the term “comprising” refers to variouscomponents conjointly employed in the manufacture and use of thecompounds and methods described herein. Accordingly, the terms“consisting essentially of” and “consisting of” are embodied in the term“comprising”.

As generally used herein, the grammatical articles including “one”, “a”,“an”, and “the” refer to “at least one” or “one or more” of what isclaimed or described, unless otherwise indicated. Thus, the articles areused herein to refer to one or more than one (i.e., to at least one) ofthe grammatical objects of the article. By way of example, “a component”means one or more components, and thus, possibly, more than onecomponent is contemplated and may be employed or used.

As generally used herein, the terms “include”, “includes” and“including” are meant to be non-limiting.

As generally used herein, the terms “have”, “has” and “having” are meantto be non-limiting.

All numerical quantities or characteristics stated herein areapproximate unless otherwise indicated, meaning that all numericalquantities are to be understood as being prefaced and modified in allinstances by the term “about”. Each numerical quantity is intended tomean both the recited value and a functionally equivalent rangesurrounding that value unless otherwise indicated. At the very least,and not as an attempt to limit the application of the doctrine ofequivalents to the scope of the claims, each numerical parameterdescribed in the present description should at least be construed inlight of the number of reported significant digits and by applyingordinary rounding techniques. Notwithstanding the approximations ofnumerical quantities stated herein, the numerical quantities describedin the Examples are reported as precisely as possible.

All numerical ranges stated herein include all sub-ranges subsumedtherein. For example, a range of “1 to 10” is intended to include allsub-ranges between and including the recited minimum value of 1 and therecited maximum value of 10, that is, having a minimum value equal to orgreater than 1 and a maximum value of equal to or less than 10. Anymaximum numerical limitation recited herein is intended to include alllower numerical limitations. Any minimum numerical limitation recitedherein is intended to include all higher numerical limitations.Accordingly, Applicant(s) reserves the right to amend the presentdisclosure, including the claims, to expressly recite any sub-rangesubsumed within the ranges expressly recited herein. All such ranges areintended to be inherently disclosed herein such that amending toexpressly recite any such sub-ranges would comply with the requirementsof 35 U.S.C. §112, first paragraph, and 35 U.S.C. §132(a).

As generally used herein, the terms “detergent” and “surfactant” referto compounds and compositions that may facilitate the solubilization ofproteins, other hydrophobic molecules, or molecules with significanthydrophobic regions in an aqueous environment.

As generally used herein, the term “cleave” refers to reducing ordestroying the detergent properties of the surfactant. In at least oneembodiment, the term “cleave” refers to separating the cleavable linkerand the polar group and/or non-polar groups. In at least one embodiment,the term “cleave” refers to degrading or disrupting the bond between thecleavable linker and the polar group and/or non-polar groups.

As generally used herein, the term “labile” refers to the property of amolecule or bond to undergo chemical, physical, or biological change,degradation, or disruption.

As generally used herein, the term “sample-surfactant complex” refers tothe molecular complex that may be formed by a surfactant and a sample.

As generally used herein, the term “sample” refers to any molecule thatmay be used with the anionic acid-labile surfactants or methodsdescribed herein, such as, for example, but not limited to, hydrophobicmolecules, molecules with significant hydrophobic regions, proteins,peptides, polypeptides, polymers, nucleic acids, lipids, lipophilliccellular components, hydrophilic extracellular components, and anycombinations thereof.

As generally used herein, when any variable occurs more than one time ina chemical formula, its definition on each occurrence is independent ofits definition at every other occurrence.

As generally used herein, a dash (“-”) that is not between two lettersor symbols is used to indicate a point of attachment for a substituent.

As generally used herein, the term “alkyl” refers to saturated aliphaticgroups, including straight-chain alkyl, branched-chain alkyl, straightor branched chain heteroalkyl, cycloalkyl, heterocyclic alkyl, alkylsubstituted cycloalkyl groups, and cycloalkyl substituted alkyl groups,including, for example, but not limited to, methyl, ethyl, n-propyl,iso-propyl, n-butyl, sec-butyl, t-butyl, pentyl, n-hexyl, andcyclohexyl.

As generally used herein, the notation “n” in reference to an organicgroup, wherein n is an integer or an integer range, indicates that thegroup may contain n carbon atoms or that range of carbon atoms pergroup. The terminology “C_(n)-C_(m)” in reference to an organic group,wherein n and m are each integers, indicates that the group may containfrom n carbon atoms to m carbon atoms per group.

Unless otherwise indicated, all compound or composition levels refer tothe active portion of that compound or composition, and are exclusive ofimpurities, for example, residual solvents or by-products, which may bepresent in commercially available sources of any compounds orcompositions.

All percentages and ratios are calculated by weight unless otherwiseindicated. All percentages and ratios are calculated based on the totalweight of the compound or composition unless otherwise indicated.

This disclosure describes various elements, features, aspects, andadvantages of various non-limiting embodiments of anionic acid-labilesurfactants and methods of use. It is to be understood that certaindescriptions of the disclosed embodiments have been simplified toillustrate only those elements, features and aspects that are relevantto a clear understanding of the disclosed embodiments, whileeliminating, for purposes of clarity, other elements, features andaspects. Persons having ordinary skill in the art, upon considering thepresent description of the disclosed embodiments, will recognize thatvarious combinations or sub-combinations of the disclosed embodimentsand other elements, features, and/or aspects may be desirable in aparticular implementation or application of the disclosed embodiments.However, because such other elements and/or features may be readilyascertained by persons having ordinary skill upon considering thepresent description of the disclosed embodiments, and are not necessaryfor a complete understanding of the disclosed embodiments, a descriptionof such elements and/or features is not provided herein. As such, it isto be understood that the description set forth herein is merelyexemplary and illustrative of the disclosed embodiments and is notintended to limit the scope of the invention as defined solely by theclaims.

B. Overview

In certain embodiments, the ionic acid-labile surfactants may generallycomprise a non-polar (hydrophobic) group joined by a cleavable linker toa polar (hydrophilic) group. In certain embodiments, the anionicacid-labile surfactants described herein may comprise two shorter chainhydrophobic tails that individually bind weaker than conventionalsurfactants, but collectively bind nearly as well. In at least oneembodiment, the interaction between the cleavable linker and the polargroup and/or non-polar group may be covalent bonding, ionic bonding,hydrogen bonding, or van der Waals bonding. In at least one embodiment,the ionic acid-labile surfactant may be cleavable. In at least oneembodiment, the ionic acid-labile surfactant may be labile. In at leastone embodiment, the ionic acid-labile surfactant may be acid cleavable,i.e., acidic conditions may be used to cleave the bond between thecleavable linker and the polar group and/or non-polar groups. In atleast one embodiment, the ionic acid-labile surfactant may be acidcleavable with the proviso that the acid is not a strong acid.

In at least one embodiment, the ionic acid-labile surfactants may behydrolyzed at a relatively low pH to generate cleavage by-products,including an ionic, water-soluble or partially water-soluble compound(e.g., an anionic head group) and a neutral, water-soluble or partiallywater-soluble compound (e.g., short to mid-length alcohols, such aspentanol, hexanol, heptanol, and octanol). These cleavage by-productsmay be removed from the sample-surfactant complex more readily than theoriginal surfactants because they exhibit reduced, if any, detergentcharacteristics and/or do not readily bind to the sample. In at leastone embodiment, the cleavage by-products may be washed away by utilizinga solid phase extraction step in which the sample may be bound to thesurface of a reversed phase chromatographic bead.

In at least one embodiment, the polar group and/or non-polar groups mayimprove the formation of a surfactant-sample complex. In at least oneembodiment, the polar group and/or non-polar groups may improve thesolubility of the cleavage by-products. In at least one embodiment, thecleavage by-products may minimize signal suppression. In at least oneembodiment, the cleavage by-products may have reduced or negligibledetergent characteristics. In at least one embodiment, the cleavageby-products may be removable by standard isolation techniques. In atleast one embodiment, fewer adducts of the sample and non-degradedsurfactant may be formed.

The anionic acid-labile surfactants described herein may be especiallyuseful for purification and identification techniques in whichconventional cleavage by-products interfere with the purification andidentification of the sample. Examples of proteomic purification andidentification technologies that may benefit from the anionicacid-labile surfactants described herein include, but are not limitedto, ion-pair liquid chromatography, liquid chromatography, massspectrometric detection, liquid-liquid extraction, solid phaseextraction, cell lysis, and other technologies that may benefit from theremoval of the surfactants after use.

C. Anionic Acid-Labile Surfactants

In certain embodiments, the ionic acid-cleavable surfactant maygenerally comprise an anionic acid-cleavable surfactant comprising atleast one non-polar group selected from the group consisting ofhydrogen, C₁-C₁₂ alkyl, and substituted C₁-C₁₂ alkyl; a polar groupcomprising an anionic group; and a cleavable linker comprising a ketalor an acetal.

In certain embodiments, the anionic acid-cleavable surfactant maygenerally comprise a compound of Formula I or a compound of Formula II:

wherein R₁ may be independently selected from the group consisting ofhydrogen, C₁-C₁₂ alkyl, substituted C₁-C₁₂ alkyl, R₂ may be selectedfrom the group consisting of hydrogen, C₁-C₁₂ alkyl, substituted C₁-C₁₂alkyl, Linker may be selected from the group consisting of C₁-C₁₂ alkyland substituted C₁-C₁₂ alkyl, X may be a cation, and Y may be an anion.In at least one embodiment, X may be selected from the group consistingof sodium, potassium, lithium, ammonium, and calcium ions. In at leastone embodiment, Y may comprise a hard anionic charge selected from thegroup consisting of sulfate, sulfonate, phosphate, phosphonate andborate ions. In at least one embodiment, Y may comprise a weak anioniccharge selected from the group consisting of carbonate, acetate, andformate ions.

In at least one embodiment, the R₁ substituted C₁-C₁₂ alkyl may beselected from the group consisting of halogen substitution (e.g., —F,—Cl, —Br, or —I substitution), heterocyclic substitution, cyclic alkylsubstitution, amide substitution, amine substitution, estersubstitution, ether substitution, and phenyl substitution. In at leastone embodiment, R₁ substituted C₁-C₁₂ alkyl may be at least one offluoralkyl substitution, pre-fluoroalkyl substitution, and benzenesubstitution.

In at least one embodiment, the R₂ substituted C₁-C₁₂ alkyl may beselected from the group consisting of alkoxy substitution and halogensubstitution (e.g., —F, —Cl, —Br, or —I substitution). In at least oneembodiment, R₂ substituted C₁-C₁₂ alkyl may be at least one offluoralkyl substitution and pre-fluoroalkyl substitution.

In at least one embodiment, the Linker substituted C₁-C₁₂ alkyl may beselected from the group consisting of halogen substitution (e.g., —F,—Cl, —Br, or —I substitution), heterocyclic substitution, cyclic alkylsubstitution, amide substitution, amine substitution, estersubstitution, ether substitution, and phenyl substitution. In at leastone embodiment, Linker substituted C₁-C₁₂ alkyl may be at least one offluoralkyl substitution, pre-fluoroalkyl substitution, and benzenesubstitution.

In certain embodiments, anionic acid-labile surfactants may generallycomprise a compound represented by Formula III or a compound representedby Formula IV:

wherein R₁ may be independently selected from the group consisting ofC₁-C₁₂ alkyl and substituted C₁-C₁₂ alkyl, R₂ may be selected from thegroup consisting of hydrogen, C₁-C₁₂ alkyl, substituted C₁-C₁₂ alkyl, Xmay be a cation, Y may be an anion, and n may be an integer from 1 to12. In at least one embodiment, R₁ may be independently selected from—(CH₂)₀₋₉CH₃, R₂ may be selected from the group consisting of —H and—(CH₂)₀₋₅CH₃, Y may be selected from the group consisting of —SO₃ ⁻ and—PO₃ ⁻, X may be a cation, and n may be an integer from 1 to 8. In atleast one embodiment, R₁ may be independently selected from—(CH₂)₀₋₉CH₃, with the proviso that R₁ does not comprise abranched-chain alkyl group or a cycloalkyl group. In at least oneembodiment, R₁ may be independently a saturated, straight-chain C₁-C₁₂alkyl group. In at least one embodiment, X may be selected from thegroup consisting of sodium, potassium, lithium, and ammonium ions.

In certain embodiments, anionic acid-labile surfactants may generallycomprise a compound represented by Formula V or compound represented byFormula VI

wherein X may be a cation and Y may be an anion. In at least oneembodiment, X may be selected from the group consisting of sodium,potassium, lithium, and ammonium ions. In at least one embodiment, Y maybe selected from the group consisting of —SO₃ ⁻ and —PO₃ ⁻. In at leastone embodiment, the anionic acid-labile surfactant may comprise sodium2,2-bis(hexyloxy)propyl sulfonate. In at least one embodiment, theanionic acid-labile surfactant may comprise sodium2,2-bis(hexyloxy)propyl sulfate.

In certain embodiments, anionic acid-labile surfactants may generallycomprise a compound represented by Formula VII or compound representedby Formula VIII

wherein X may be a cation and Y may be an anion. In at least oneembodiment, X may be selected from the group consisting of sodium,potassium, lithium, and ammonium ions. In at least one embodiment, Y maybe selected from the group consisting of —SO₃ ⁻ and —PO₃ ⁻. In at leastone embodiment, the anionic acid-labile surfactant may comprise sodium2,2-bis(heptyloxy)propyl sulfonate. In at least one embodiment, theanionic acid-labile surfactant may comprise sodium2,2-bis(heptyloxy)propyl sulfate.

In certain embodiments, anionic acid-labile surfactants may generallycomprise a compound represented by Formula III or compound representedby Formula IV having a rate of degradation of less than 30 minutes. Inat least one embodiment, the rate of degradation may be 4-24 minutes. Inat least one embodiment, the rate of degradation may be 6-12 minutes. Inat least one embodiment, the rate of degradation may be 10 minutes. Therate of degradation relates to the rate of how easily the surfactantdegrades. Without wishing to be bound to any particular theory, the rateof degradation may depend on the stability of the sample-surfactantcomplex. The stability of the sample-surfactant complex may depend onthe chemical structure of the surfactant and/or the chemical structureof the sample-surfactant complex. For example, the rate of degradationmay depend on the electron donating groups and/or electron withdrawinggroups.

In certain embodiments, a composition may comprise a sample-surfactantcomplex. In at least one embodiment, the sample-surfactant complex maygenerally comprise a sample and an anionic acid-labile surfactantaccording to Formula III or Formula IV. In at least one embodiment, thecomposition may comprise an anionic acid-labile surfactant according toFormula III or Formula IV and a protein mixture for electrophoresis.Without wishing to be bound to any particular theory, in solution, thehydrophobic tails of the surfactants may associate with the hydrophobicportion of the sample, e.g., proteins, via hydrophobic interactions. Thehydrophilic heads of the surfactants may align outwardly from thehydrophobic tails to maximize the distance between the two opposingchemistries, and toward the bulk aqueous solvent where the hydrophilicheads may associate with the polar water molecules. In at least oneembodiment, the sample-surfactant complex may improve the solubility ofthe native (uncomplexed) sample. In at least one embodiment, thesample-complex may improve the solubility of the native sample in thatthe hydrophilic heads provide a cumulative improvement in the solublenature of the sample-surfactant complex. In at least one embodiment, thesample-surfactant complex may provide an increased potential forsolvation and maintenance of a dissolved state.

D. Synthesis of Certain Embodiments

The synthesis of the anionic acid-labile surfactant compounds may becarried out using commercially available starting materials and knownchemical reaction steps. For example, synthetic methods for thepreparation of various surfactant compounds may be described in Huibers,M.; Manuzi, A.; Rutjes, F.; Delft, F. J. Org. Chem., 2006, 71,7473-7476; Ono, D.; Yamamura, S.; Nakamura, M.; Takeda, T. J. Ole. Sci.,2004, 53 (2), 89-95; and Guo, W.; Li, Z.; Fung, B. M. J. Phys. Chem.,1992, 96, 6738-6742. The methods of synthesizing the anionic acid-labilesurfactants may produce isomers. Although the methods of using theanionic acid-labile surfactants may not require separation of theseisomers, such separation may be accomplished, if desired, by standardseparation methods, such as, for example, preparative high performanceliquid chromatography.

The following examples for the preparation of anionic acid-labilesurfactants are for illustrative purposes, and not intended to limit thescope of the anionic acid-labile surfactants compounds and methodsdescribed herein. Additionally, in practicing the anionic acid-labilesurfactants and methods, one of ordinary skill in the art wouldunderstand that various modifications to the following procedures wouldbe routine, in light of the teachings herein, and that suchmodifications would be within the spirit and scope of the anionicacid-labile surfactants compounds and methods described herein.

1. Preparation of sodium 2,2-bis(hexyloxy)propyl sulfate

¹H NMR and ¹³C NMR spectra are recorded on a Varian 600 MHzspectrometer. Chemical shifts are reported relative to CDCl₃ (δ 7.24ppm) or C₆D₆ (δ 7.16 ppm) for ¹H NMR and CDCl₃ (δ 77.0 ppm) or C₆D₆ (δ128.4 ppm) for ¹³C NMR Infrared (IR) spectra are obtained on a FT-IRspectrometer. Sorbtech 60A (230-400 mesh) silica gel is used for flashchromatography. Analytical thin-layer chromatography is performed withprecoated glass-backed plates (K6F 60 Å, F254) and visualized byquenching of fluorescence and by charring after treatment withp-anisaldehyde or phosphomolybdic acid or potassium permanganate stain.R_(f) values are obtained by elution in the stated solvent ratios (v/v).Ether (Et₂O), methylene chloride (CH₂Cl₂) and toluene are dried bypassing through an activated alumina (8×14 mesh) column with argon gaspressure. Commercial reagents are purchased from Fisher Scientific orSigma-Aldrich and used without purification unless otherwise noted. Airand/or moisture-sensitive reactions are carried out under an atmosphereof argon/nitrogen using oven/flamed-dried glassware and standardsyringe/septa techniques.

a. Synthesis of hexyl 2,2-bis(hexyloxy)propanoate

To a solution of methyl pyruvate 1 (10.0 g, 98.0 mmol) in toluene (100mL) is added 1-hexanol 2 (40.1 g, 392 mmol) and p-Ts0H (186 mg, 0.98mmol). The mixture is heated to reflux for 10 hours with azeotropicremoval of water from the reaction mixture. The reaction is quenchedwith saturated NaHCO₃ (100 mL), and the reaction mixture is extractedwith ethyl acetate (2×100 mL). The combined organic layers are washedwith brine (100 mL) and dried over anhydrous sodium sulfate. The solventis removed and the residue is purified by silica gel chromatography(1→10% ethyl acetate/hexane) to give hexyl 2,2-bis(hexyloxy)propanoate 3(29.5 g, 84%) as a colorless oil: R_(f) (15% EtOAc/hexane)=0.53; IR(thin film, cm⁻¹) 2956, 2930, 2860, 1746 (C═O), 1467, 1280, 1137, 1062.¹H NMR (600 MHz, CDCl₃) δ 4.14 (t, J=7.2 Hz, 2H), 3.48 (ddd, J=9.0, 7.2,6.6 Hz, 2H), 3.35 (ddd, J=9.0, 7.2, 6.6 Hz, 2H), 1.65-1.63 (m, 2H),1.59-1.54 (m, 4H), 1.49 (s, 3H), 1.35-1.24 (m, 18H), 0.86 (t, J=7.2 Hz,9H); ¹³C NMR (150 MHz, CDCl₃) δ 170.2, 99.5, 65.4, 62.6, 31.7, 31.3,29.7, 28.5, 25.8, 25.5, 22.6, 22.5, 21.9, 14.0, 13.9.

b. Synthesis of 2,2-bis(hexyloxy)propan-1-ol

To a mixture of LiAlH₄ (3.44 g, 90.5 mmol) in Et₂O (200 mL) is added asolution of ester 3 (29.5 g, 82.3 mmol) in Et₂O (100 mL). Afteraddition, the mixture is refluxed for 6 hours. The reaction mixture iscooled to 0° C. and quenched with ethyl acetate (20 mL) and H₂O (20 mL).The mixture is added to saturated potassium sodium tartrate (300 mL) andstirred at 23° C. for 12 hours. The mixture is extracted with Et₂O(2×200 mL) and the combined organic layers are dried over anhydroussodium sulfate. The solvent is removed and the residue is purified bysilica gel chromatography (1→10% ethyl acetate/hexane) to give2,2-bis(hexyloxy)propan-1-ol 4 (20.6 g, 94%) as a colorless oil: R_(f)(15% EtOAc/hexane)=0.20; IR (thin film, cm⁻¹) 3430, 2955, 2929, 2859,1467, 1378, 1244, 1155, 1112, 1064, 876; ¹H NMR (600 MHz, C₆D₆) δ 3.58(d, J=6.6 Hz, 2H), 3.43 (t, J=6.6 Hz, 4H), 1.58 (t, J=6.6 Hz, 1H), 1.53(m, 4H), 1.36 (s, 3H), 1.34-1.22 (m, 12H), 0.88 (t, J=7.2 Hz, 6H); ¹³CNMR (150 MHz, C₆D₆) δ 101.0, 66.2, 61.3, 32.5, 30.9, 26.8, 23.4, 21.3,14.6.

c. Synthesis of sodium 2,2-bis(hexyloxy)propyl sulfate

To a solution of alcohol (2.50 g, 9.60 mmol) in pyridine (5 mL) is addedSO₃.Py (2.29 g, 14.4 mmol). The reaction mixture is stirred at 80° C.for 10 hours. The reaction mixture is cooled to 23° C. and quenched bypouring the mixture into an ice-cooled sodium carbonate solution (10 gin 20 mL of water). The mixture is extracted with butanol (2×50 mL). Thecombined organic layers are washed with brine and dried over anhydroussodium sulfate. The solvent is removed and the residue is purified bysilica gel chromatography (10→100% ethyl acetate/hexane) to give sodium2,2-bis(hexyloxy)propanyl ethyl sulfate 5 (4.07 g, 74%) as a colorlesssolid: R_(f) (20% EtOH/EtOAc)=0.48; IR (thin film, cm⁻¹) 3506, 2956,2930, 2056, 1642, 1467, 1380, 1228, 1015, 820; ¹H NMR (600 MHz, CDCl₃) δ3.99 (s, 2H), 3.42 (t, J=7.2 Hz, 4H), 1.51-1.49 (m, 4H), 1.36 (s, 3H),1.30-1.24 (m, 12H), 0.86 (t, J=7.2 Hz, 6H); ¹³C NMR (150 MHz, CDCl₃) δ99.2, 69.8, 60.8, 31.8, 29.8, 25.9, 22.7, 20.8, 14.0.

2. Preparation of sodium 2,2-bis(hexyloxy)propyl sulfate

¹H NMR and ¹³C NMR spectra are recorded on a Varian 600 MHzspectrometer. Chemical shifts are reported relative to CDCl₃ (δ 7.24ppm) or C₆D₆ (δ 7.16 ppm) for ¹H NMR and CDCl₃ (δ 77.0 ppm) or C₆D₆ (δ128.4 ppm) for ¹³C NMR. Infrared (IR) spectra are obtained on a FT-IRspectrometer. Sorbtech 60A (230-400 mesh) silica gel is used for flashchromatography. Analytical thin-layer chromatography is performed withprecoated glass-backed plates (K6F 60 Å, F254) and visualized byquenching of fluorescence and by charring after treatment withp-anisaldehyde or phosphomolybdic acid or potassium permanganate stain.R_(f) values are obtained by elution in the stated solvent ratios (v/v).Ether (Et₂O), methylene chloride (CH₂Cl₂) and toluene are dried bypassing through an activated alumina (8×14 mesh) column with argon gaspressure. Commercial reagents are purchased from Fisher Scientific orSigma-Aldrich and used without purification unless otherwise noted. Airand/or moisture-sensitive reactions are carried out under an atmosphereof argon/nitrogen using oven/flamed-dried glassware and standardsyringe/septa techniques.

a. Synthesis of hexyl 2,2-bis(hexyloxy)propanoate

To a solution of methyl pyruvate 1 (10.0 g, 98.0 mmol) in toluene (100mL) is added 1-hexanol 2 (40.1 g, 392 mmol) and p-Ts0H (186 mg, 0.98mmol). The mixture is heated to reflux for 10 hours with azeotropicremoval of water from the reaction mixture. The reaction is quenchedwith saturated NaHCO₃ (100 mL), and the reaction mixture is extractedwith ethyl acetate (2×100 mL). The combined organic layers are washedwith brine (100 mL) and dried over anhydrous sodium sulfate. The solventis removed and the residue is purified by silica gel chromatography(1→10% ethyl acetate/hexane) to give hexyl 2,2-bis(hexyloxy)propanoate 3(29.5 g, 84%) as a colorless oil: R_(f) (15% EtOAc/hexane)=0.53; IR(thin film, cm⁻¹) 2956, 2930, 2860, 1746 (C═O), 1467, 1280, 1137, 1062.¹H NMR (600 MHz, CDCl₃) δ 4.14 (t, J=7.2 Hz, 2H), 3.48 (ddd, J=9.0, 7.2,6.6 Hz, 2H), 3.35 (ddd, J=9.0, 7.2, 6.6 Hz, 2H), 1.65-1.63 (m, 2H),1.59-1.54 (m, 4H), 1.49 (s, 3H), 1.35-1.24 (m, 18H), 0.86 (t, J=7.2 Hz,9H); ¹³C NMR (150 MHz, CDCl₃) δ 170.2, 99.5, 65.4, 62.6, 31.7, 31.3,29.7, 28.5, 25.8, 25.5, 22.6, 22.5, 21.9, 14.0, 13.9.

b. Synthesis of 2,2-bis(hexyloxy)propan-1-ol

To a mixture of LiAlH₄ (3.44 g, 90.5 mmol) in Et₂O (200 mL) is added asolution of ester 3 (29.5 g, 82.3 mmol) in Et₂O (100 mL). Afteraddition, the mixture is refluxed for 6 hours. The reaction mixture iscooled to 0° C. and quenched with ethyl acetate (20 mL) and H₂O (20 mL).The mixture is added to saturated potassium sodium tartrate (300 mL) andstirred at 23° C. for 12 hours. The mixture is extracted with Et₂O(2×200 mL) and the combined organic layers are dried over anhydroussodium sulfate. The solvent is removed and the residue is purified bysilica gel chromatography (1→10% ethyl acetate/hexane) to give2,2-bis(hexyloxy)propan-1-ol 4 (20.6 g, 94%) as a colorless oil:R_(f)(15% EtOAc/hexane)=0.20; IR (thin film, cm⁻¹) 3430, 2955, 2929,2859, 1467, 1378, 1244, 1155, 1112, 1064, 876; ¹H NMR (600 MHz, C₆D₆) δ3.58 (d, J=6.6 Hz, 2H), 3.43 (t, J=6.6 Hz, 4H), 1.58 (t, J=6.6 Hz, 1H),1.53 (m, 4H), 1.36 (s, 3H), 1.34-1.22 (m, 12H), 0.88 (t, J=7.2 Hz, 6H);¹³C NMR (150 MHz, C₆D₆) δ 101.0, 66.2, 61.3, 32.5, 30.9, 26.8, 23.4,21.3, 14.6.

c. Synthesis of 2,2-bis(hexyloxy)propyl ethyl sulfite

To a solution of alcohol 4 (20.6 g, 79.1 mmol) in CH₂Cl₂ (200 mL) isadded pyridine (8.12 g, 102.8 mmol) and ethyl chlorosulfite (12.2 g,94.9 mmol). The reaction mixture is stirred at 23° C. for 10 hours. Thereaction is quenched with water (100 mL) and extracted with CH₂Cl₂(2×100 mL). The combined organic layers are washed with HCl (1N, 100mL), saturated NaHCO₃ (100 mL), brine (100 mL) and dried over anhydroussodium sulfate. The solvent is removed and the residue is purified bysilica gel chromatography (1→10% ethyl acetate/hexane) to give2,2-bis(hexyloxy)propanyl ethyl sulfite 6 (24.5 g, 88%) as a colorlessoil: R_(f) (15% EtOAc/hexane)=0.44; IR (thin film, cm⁻¹) 2932, 2872,1467, 1379, 1213, 1194, 1176, 1001, 888; ¹H NMR (600 MHz, CDCl₃) δ4.11-4.00 (m, 2H), 3.93 (d, J=10.8 Hz, 1H), 3.77 (d, J=10.8 Hz, 1H),3.42-3.37 (m, 4H), 1.51-1.48 (m, 4H), 1.33 (s, 3H), 1.31-1.24 (m, 15H),0.85 (t, J=7.2 Hz, 6H); ¹³C NMR (150 MHz, CDCl₃) δ 99.0, 62.9, 60.8,58.5, 58.3, 31.6, 29.8, 25.9, 22.6, 20.9, 15.3, 14.0.

d. Synthesis of 2,2-bis(hexyloxy)propyl ethyl sulfate

To a solution of sulfite 6 (24.5 g, 69.5 mmol) in MeCN (200 mL), CH₂Cl₂(200 mL) and water (300 mL) is added NaIO₄ (29.7 g, 139.0 mmol) andRuCl₃ (10 mg, 0.05 mmol). The mixture is stirred at 23° C. for 5 hours.The mixture is filtered through Celite and extracted with CH₂Cl₂ (2×200mL). The combined organic layers are washed with saturated NaHCO₃ (100mL), brine (100 mL) and dried over anhydrous sodium sulfate. The solventis removed and the residue is purified by silica gel chromatography(1→10% ethyl acetate/hexane) to give 2,2-bis(hexyloxy)propanyl ethylsulfate 7 (23.3 g, 91%) as a colorless oil: R_(f) (15%EtOAc/hexane)=0.41; IR (thin film, cm⁻¹) 2931, 2860, 1737, 1467, 1403,1380, 1196, 1179, 1012, 925, 858; ¹H NMR (600 MHz, CDCl₃) δ 4.34 (q,J=7.2 Hz, 2H), 4.09 (s, 2H), 3.43 (ddd, J=9.0, 7.2, 6.6 Hz, 2H), 3.38(ddd, J=9.0, 7.2, 6.6 Hz, 2H), 1.51 (m, 4H), 1.40 (t, J=7.2 Hz, 3H),1.37 (s, 3H), 1.34-1.24 (m, 12H), 0.86 (t, J=7.2 Hz, 6H); ¹³C NMR (150MHz, CDCl₃) δ 98.2, 72.4, 69.7, 60.9, 31.6, 29.8, 25.9, 22.6, 20.8,14.5, 14.0.

e. Synthesis of Sodium 2,2-bis(hexyloxy)propyl sulfate

To a solution of sulfate diester 7 (5.6 g, 15.2 mmol) in acetone (15 mL)is added NaI (4.56 g, 30.4 mmol). The solution is stirred at 23° C. for10 hours. The solvent is removed and the residue is purified by silicagel chromatography (10→100% ethyl acetate/hexane) to give sodium2,2-bis(hexyloxy)propanyl ethyl sulfate 8 (4.07 g, 74%) as a colorlesssolid: R_(f) (20% EtOH/EtOAc)=0.48; IR (thin film, cm⁻¹) 3506, 2956,2930, 2056, 1642, 1467, 1380, 1228, 1015, 820; ¹H NMR (600 MHz, CDCl₃) δ3.99 (s, 2H), 3.42 (t, J=7.2 Hz, 4H), 1.51-1.49 (m, 4H), 1.36 (s, 3H),1.30-1.24 (m, 12H), 0.86 (t, J=7.2 Hz, 6H); ¹³C NMR (150 MHz, CDCl₃) δ99.2, 69.8, 60.8, 31.8, 29.8, 25.9, 22.7, 20.8, 14.0.

E. Uses for Certain Compounds

In certain embodiments, the anionic acid-labile surfactants may be usedto facilitate the solubilization of proteins and other molecules in anaqueous environment. In at least one embodiment, the anionic acid-labilesurfactants may be used in purification and identification technologiesincluding, but are not limited to, ion-pair liquid chromatography,liquid chromatography, mass spectrometry (ESI and MALDI MS),liquid-liquid extraction, solid phase extraction, HPLC/MS, HPLC/UVanalyses and other techniques that benefit from the removal of theanionic surfactant after use. In at least one embodiment, the anionicacid-labile surfactants may be used in electrophoresis, capillaryelectrophoresis, electroelution, cell lysis and protein extraction fromcell lines, tissues, and biological samples, selective proteinextraction from biological samples, extraction of biomolecules fromenvironmental samples, enzymatic digestion of proteins, disruption ofprotein-protein interactions, and protein denaturation.

In certain embodiments, a method of isolating a sample may generallycomprise adjusting the sample to pH 6-12; mixing a solvent and ananionic acid-labile surfactant according to Formula III or Formula IV;contacting the sample with the mixture to form a sample-surfactantcomplex; cleaving the surfactant from the sample-surfactant complex toform cleavage by-products; and isolating the sample from the cleavageby-products. In at least one embodiment, the method of isolating asample may comprise agitating at least one of the sample, mixture, andsample-surfactant complex. In at least one embodiment, agitating maycomprise sonication. In at least one embodiment, the method of isolatinga sample may comprise sonicating the sample-surfactant complex. In atleast one embodiment, the method of isolating a sample may compriseperforming mass spectrometry on the isolated sample. In at least oneembodiment, the cleavage by-products may be soluble in at least one ofthe cleaved sample-surfactant complex. In at least one embodiment, thecleavage by-products may be soluble in the isolated sample.

In at least one embodiment, the solvent may be selected from the groupconsisting of water; 0-50% methanol; 0-50% acetonitrile; 5-50 mMammonium bicarbonate buffer; 5-50 mM Tris-HCl buffer; 5-50 mM sodiumphosphate buffer, 5-50 mM ammonium acetate buffer, and any combinationthereof.

In at least one embodiment, adjusting the sample to pH 6-12 may comprisecontacting the sample with an acid or a base. In at least oneembodiment, adjusting the sample to pH 6-12 may comprise contacting thesample with an acid with the proviso that the acid is not a strong acid.In at least one embodiment, adjusting the sample to pH 6-12 may comprisecontacting the sample with a weak acid. In at least one embodiment, theacid may be selected from the group consisting of formic acid, aceticacid, trifluoroacetic acid, heptafluorobutyric acid, citric acid,phosphoric acid, and boric acid. In at least one embodiment, the basemay be selected from the group consisting of ammonium hydroxide, sodiumhydroxide, and potassium hydroxide.

In at least one embodiment, cleaving may comprise adjusting thesample-surfactant complex to pH 2-3. In at least one embodiment,adjusting to pH 2-3 may comprise contacting the sample-surfactantcomplex with an acid. In at least one embodiment, the adjusting to pH2-3 may comprise contacting the sample-surfactant complex with an acidwith the proviso that the acid in not a strong acid. In at least oneembodiment, adjusting to pH 2-3 may comprise contacting thesample-surfactant complex with a weak acid. In at least one embodiment,the acid may be selected from the group consisting of formic acid,trifluoroacetic acid, heptafluorobutyric acid, and acetic acid.

In at least one embodiment, cleaving may comprise incubating thesample-surfactant complex. In at least one embodiment, cleaving maycomprise incubating the sample-surfactant complex for less than four (4)hours at less than 99° C. In at least one embodiment, cleaving maycomprise incubating the sample-surfactant complex from 5 minutes to 1(one) hour. In at least one embodiment, cleaving may comprise incubatingthe sample-surfactant complex from 4-50° C. In at least one embodiment,cleaving may comprise incubating the sample-surfactant complex from 4°C. to room temperature. In at least one embodiment, cleaving maycomprise incubating the sample-surfactant complex for 10-30 minutes at4-50° C. In at least one embodiment, cleaving may comprise incubatingfor 10 minutes at room temperature.

In certain embodiments, isolating the sample from the cleavageby-products may comprise performing purification and/or identificationtechnologies. In at least one embodiment, isolating the sample from thecleavage by-products may comprise at least one of reversed phase sampleclean-up techniques and solid phase extraction techniques. In at leastone embodiment, isolating the sample from the cleavage by-products maybe selected from the group consisting of ion exchange, hydrophilicinteraction, reversed phase chromatographic preparations, and anycombination thereof.

In certain embodiments the method of isolating a sample may compriseperforming electrokinetic transport of the sample-surfactant complex. Inat least one embodiment, performing electrokinetic transport maycomprise electrophoresis. In at least one embodiment, electrophoresismay comprise gel electrophoresis, free zone electrophoresis, andcapillary electrophoresis. In at least one embodiment, electrophoresismay comprise polyacrylamide gel electrophoresis, including the tube,slab gel and capillary formats of polyacrylamide gel electrophoresis.

In certain embodiments the method of isolating a sample may comprisepurifying the isolated sample. In at least one embodiment, purifying maycomprise conventional separation methods, including, but not limited to,liquid-liquid extraction, solid-phase extraction and liquidchromatography.

In certain embodiments the method of isolating a sample may compriseperforming enzymatic digestion of the sample-surfactant complex. In atleast one embodiment, performing enzymatic digestion may compriseforming a sample-surfactant complex by contacting a sample and anacid-labile surfactant according to Formula III or Formula IV (finalconcentration of 0.01-1.0%) in a buffered solution of 10-100 mM ammoniumbicarbonate; incubating the sample-surfactant complex withdithiothreitol (DTT) (5-50 mM) for less than one (1) hour at 50-60° C.for reduction of cysteine-cysteine disulfide linkages; cooling thesample-surfactant complex mixture; incubating the sample-surfactantcomplex mixture with iodoacetamide (25-250 mM) for less than one (1)hour at less than 30° C. in limited light; mixing the sample-surfactantcomplex with an enzyme; and incubating the mixture for less than 24hours at 20-40° C. with shaking. In at least one embodiment, performingenzymatic digestion may comprise incubating the sample-surfactantcomplex in 50 mM ammonium bicarbonate for 30 minutes at 55° C. with 5 mMDTT; cooling the sample-surfactant complex to room temperature;incubating the sample-surfactant complex for 30 minutes at roomtemperature in the dark in 25 mM iodoacetamide; mixing thesample-surfactant complex with an enzyme; and incubating the mixture for4-12 hours at 37° C. with shaking. In at least one embodiment, theenzyme may be selected from the group consisting of trypsin, Glu-C,Arg-C, Lys-C, Asp-N, chymotrypsin, and pepsin.

In at least one embodiment, performing enzymatic digestion may compriseincubating the sample-surfactant complex for less than one (1) hour atless than 99° C.; cooling the sample-surfactant complex; incubating thesample-surfactant complex for less than one (1) hour at less than 99° C.in limited light; mixing the sample-surfactant complex with an enzyme;and incubating the mixture for less than 12 hours at 4-55° C. withshaking. In at least one embodiment, performing enzymatic digestion maycomprise incubating the sample-surfactant complex for 30 minutes at 55°C.; cooling the sample-surfactant complex to room temperature;incubating the sample-surfactant complex for 30 minutes at roomtemperature in the dark; mixing the sample-surfactant complex with anenzyme; and incubating the mixture for 4-8 hours at 37° C. with shaking.

In certain embodiments the method of isolating a sample may furthercomprise desalting the sample-surfactant complex with an enzyme. In atleast one embodiment, desalting may comprise cleaving thesample-surfactant complex. In at least one embodiment, desalting maycomprise degrading the surfactant of the sample-surfactant complex. Inat least one embodiment, desalting may comprise loading the mixture ofthe sample and degraded surfactant onto a solid phase extractionchromatographic media; washing away the salts and surfactant degradationproducts, and collecting the sample from the solid phase extractionmedia by elution. In at least one embodiment, the solid phase extractionmedia may be selected from the group consisting of reversed phase, ionexchange, hydrophilic interaction (HILIC), and any combination thereof.

In certain embodiments, a method for analyzing a sample may generallycomprise contacting a sample with an anionic acid-labile surfactantaccording to Formula III or Formula IV to form a sample-surfactantcomplex and analyzing the sample-surfactant complex. In at least oneembodiment, analyzing may comprise purification and/or identificationtechnologies. In at least one embodiment, analyzing may comprise atleast one of electrophoresis, electroelution, high performance liquidchromatography, mass spectrometric detection, liquid-liquid extraction,solid phase extraction, and ion-pair liquid chromatography. In at leastone embodiment, the method for analyzing a sample may generally comprisedegrading the surfactant. In at least one embodiment, the method foranalyzing a sample may generally comprise purifying the sample afterdegrading the surfactant.

In certain embodiments, a method for performing electrophoresis maygenerally comprise contacting a sample with an anionic acid-labilesurfactant according to Formula III or Formula IV to form asample-surfactant complex, performing electrophoresis on thesample-surfactant complex, and degrading the surfactant afterelectrophoresis. In at least one embodiment, degrading may comprisecontacting the surfactant with an acidic solution. In at least oneembodiment, degrading may comprise contacting the surfactant with anacid with the proviso that the acid is not a strong acid. In at leastone embodiment, the method for performing electrophoresis may generallycomprise purifying the sample after degrading the surfactant.

E. Examples

The various embodiments of anionic acid-labile surfactants and methodsof use described herein may be better understood when read inconjunction with the following representative examples. The followingexamples are included for purposes of illustration and not limitation.

Comparisons of certain embodiments of anionic acid-labile surfactantsand commercially available cleavable surfactants are described.Cleavable surfactants generally exhibit various properties, including,but not limited to, lability (rate, efficiency, cleavage products),compatibility with mass spectrometry (level of signal suppression,adduct formation, artifact peaks, sample prep requirements) and otherpurification and identification technologies, such as polyacrylamide gelelectrophoresis and mass spectrometry detection, and detergent strengthand utility (CMC, ability to perform electrophoresis).

The commercially available cleavable surfactants include PPS, availablefrom Protein Discovery, Knoxyille, Tenn., Proteamax, available fromPromega, Madison, Wis., and Rapigest, available form Waters, Milford,Mass. Proteasmax and Rapigest are anionic acid labile surfactants andPPS is a zwitterionic acid labile surfactant.

1. Protein Scores from in-Solution Digestion of BSA

Protein scores from in-solution digestion of BSA in buffers containingan embodiment of an anionic acid-labile surfactant (“AALS-6”) and threecommercially available acid labile surfactants are illustrated in FIG.2. A tryptic digestion of 250 pmol BSA is performed using 0.05% AALS-6in digestion buffer. For the other surfactants, digestion is performedaccording to the manufacturer's protocols. Sample clean-up and MALDIspotting is performed using C18 reversed phase tips. MALDI-TOF spectraare acquired using an ABI 4800 mass spectrometer in the reflector mode.Protein identification is accomplished through peptide massfingerprinting (PMF) of the acquired MS spectra.

As shown in FIG. 2, AALS-6 has an average protein score of 93.Algorithms that compare mass spectrometric data to database for proteinidentification compare the experimental tandem mass spectra againsttheoretical mass spectra from all peptides and proteins present in thedatabases. As an output for the database correlation analysis, a proteinidentification score is generated for each potential protein match.Protein matches are required to have a minimum protein score to beconsidered a potential positive and significant protein identification.The higher the score, the more likely that the protein identification isreal and not a result of random events that match the database.Comparison of the protein scores for two samples that are identified asthe same protein gives an indication of the quality of the data that ledto the positive protein identification. Samples that containcontaminants or a lower amount of sample protein produce lower qualitymass spectra, which in turn may lead to lower protein ID scores. AALS-6has a higher average protein score than the other 3 commerciallyavailable surfactants, indicating that the quality of the digestedprotein sample presented to the mass spectrometer is of higher qualityfor the AALS-6 sample. The higher protein ID scores suggest that thedigestion of the BSA protein is more complete, producing a larger numberof peptides for delivery to the mass spectrometer, which produces morecomplete mass spectrometric data.

2. Protein Scores from in-Solution Digestion of Ovalbumin

The protein scores from in-solution digestion of ovalbumin in bufferscontaining an embodiment of an anionic acid-labile surfactant (“AALS-6”)and three commercially available acid labile surfactants are illustratedin FIG. 3. A tryptic digestion of 250 μmol ovalbumin was performed using0.05% AALS-6 in digestion buffer. For the other surfactants, digestionwas performed according to the manufacturer's protocols. Sample clean-upand MALDI spotting was performed using C18 reversed phase tips.MALDI-TOF MS and MS/MS spectra were acquired using an ABI 4800 massspectrometer in the reflector mode. Protein identification wasaccomplished through database searching MS/MS spectra using Mascotsearch engine.

As shown in FIG. 3, AALS-6 has an average protein score of 487 and theconventional acid labile surfactants have average protein scores of 329(ProteasMax), 341 (Rapigest), and 166 (PPS). The ovalbumin controldigestion score is 347. In general, the higher protein score indicatesan increase in the quality and/or completeness of the digestion becausemore peptides are available for analysis by the mass spectrometer,and/or the quality of the digested sample delivered to the massspectrometer is increased. Without intending to be bound by anyparticular theory, the higher protein score of AALS-6 may be due is dueto its detergent characteristics, e.g., critical micellularconcentration of 7.7 mM, which may improve both the solubility of thesample and the extent of denaturation of its 3-dimensional structure byproviding improved enzyme access to the cleavage points. The higherprotein score of AALS-6 may also be due to its degradation kinetics. Forexample, once the digestion experiment is complete, the degradation ofthe surfactant reduces contamination of the signal by detergentmolecules and by-products as well as reduces the ion suppression effectscommonly seen in mass spectrometry analysis of peptides and proteins.

3. Protein Electroelution

An embodiment of an anionic acid-labile surfactant (“AALS-6”) is usedfor protein electroelution from a polyacrylamide gel using Protea'sGPR-800 system. A gel spot comprising the protein is placed in the gelloading reservoirs. A 0.5% anionic acid-labile surfactant is prepared bymixing the anionic acid-labile surfactant with Tris-glycine buffer. Themixture is placed in the gel loading reservoir to form aprotein-surfactant complex (sample-surfactant complex). Theprotein-surfactant complex exhibits a blue color from the Coomassie bluedye molecules that are associated with the protein during the stainingprocess. The mixture is used to transfer a protein from a coomassiestained gel into solution when voltage is applied to the sample. Themovement of the protein-surfactant complex is tracked by thedisappearance of the blue color of coomassie from the gel loadingreservoirs and the emergence of the blue color of coomassie in thesample collection reservoirs. The movement of the protein-surfactant iscomplete after 15 min. The presence of the protein in the collectionreservoirs is confirmed by MALDI-TOF mass spectrometry.

Similar experiments are performed with three (3) conventional anionicacid-labile surfactants. A 0.5% Proteamax, 0.5% Rapigest, and 0.5% PPSare prepared by mixing the surfactants with Tris-glycine buffer. Noprotein is detected by MALDI-TOF mass spectrometry in the collectionreservoirs after 15 min for the 0.5% Proteamax and 0.5% Rapigest. Nomovement of the protein-coomassie complex is detectable for the 0.5% PPSsurfactant.

4. Degradation Time V. Signal-To-Noise Ratio

Referring to FIG. 1, the degradation time versus signal-to-noise ratiois compared for a certain embodiment of an anionic acid-labilesurfactant (“AALS-6”) and three commercially available acid-labilesurfactants. A 0.1% of each surfactant is mixed with 20 pmol Myoglobinand degraded for 0, 5, 10, 15, 30, 45, 60, 90, 120 and 240 min. Thesamples are spotted onto a MALDI target using C8 reversed phase tips.MALDI-TOF spectra are acquired using an ABI 4800 mass spectrometer inthe linear mode.

As shown in FIG. 1, the anionic acid-labile surfactant may improve thesensitivity of mass spectrometry analysis of proteins in the presence ofthe degraded anionic acid-labile surfactants. Cleavage of these anionicacid-labile surfactants may eliminate the detergent properties thatinduce signal suppression and/or create signal adducts in the massspectra. Adduct peaks in mass spectra may result from a population ofsurfactant-protein complexes containing different numbers of surfactantmolecules per protein, resulting in many different complexes (withdifferent masses) to be detected. If there is no surfactant present toform a surfactant-protein complex, then these adducts may not be formedor observed. This mechanism demonstrates the utility for removingsurfactants by cleavage to improve mass spectrometry analysis. Theseeffects may increase the signal intensity of analytes and eliminate thesuppressive effects of conventional detergents.

All documents cited herein are, in relevant part, incorporated herein byreference, but only to the extent that the incorporated material doesnot conflict with existing definitions, statements, or other documentsset forth herein. To the extent that any meaning or definition of a termin this document conflicts with any meaning or definition of the sameterm in a document incorporated by reference, the meaning or definitionassigned to that term in this document shall govern. The citation of anydocument is not to be construed as an admission that it is prior artwith respect to the anionic acid-labile surfactants and methods of usedescribed herein.

While particular exemplary embodiments of anionic acid-labilesurfactants and methods of use have been illustrated and described, itwould be obvious to those skilled in the art that various other changesand modifications can be made without departing from the spirit andscope of the invention. Those skilled in the art will recognize, or beable to ascertain using no more than routine experimentation, numerousequivalents to the specific devices and methods described herein,including alternatives, variants, additions, deletions, modificationsand substitutions. This disclosure, including the claims, is intended tocover all such equivalents that are within the spirit and scope of thisinvention.

1. A method of cleaving a compound of the formula:

wherein R₁ is independently selected from —(CH₂)₀₋₉CH₃, R₂ is selectedfrom the group consisting of —H and —(CH₂)₀₋₅CH₃, Y is selected from thegroup consisting of —SO₃ ⁻ and —PO₃ ⁻, X is a cation selected from thegroup consisting of sodium, potassium, lithium, and ammonium ions, and nis an integer from 1 to 8, the method comprising contacting the compoundand an acid.
 2. The method of claim 1, wherein each R₁ is —(CH₂)₅CH₃, R₂is —CH₃, and n is
 1. 3. The method of claim 1, wherein each R₁ is—(CH₂)₆CH₃, R₂ is —CH₃, and n is
 1. 4. The method of claim 1, whereineach R₁ is —(CH₂)₅CH₃, R₂ is —CH₃, Y is —SO₃ ⁻, X is sodium, and n is 1.5. The method of claim 1, wherein each R₁ is —(CH₂)₆CH₃, R₂ is —CH₃, Yis —SO₃ ⁻, X is sodium, and n is
 1. 6. A method of solubilizing asample, the method comprising contacting the sample and a compound ofthe formula:

wherein R₁ is independently selected from —(CH₂)₀₋₉CH₃, R₂ is selectedfrom the group consisting of —H and —(CH₂)₀₋₅CH₃, Y is selected from thegroup consisting of —SO₃ ⁻ and —PO₃ ⁻, X is a cation selected from thegroup consisting of sodium, potassium, lithium, and ammonium ions, and nis an integer from 1 to
 8. 7. The method of claim 6, wherein each R₁ is—(CH₂)₅CH₃, R₂ is —CH₃, and n is
 1. 8. The method of claim 6, whereineach R₁ is —(CH₂)₆CH₃, R₂ is —CH₃, and n is
 1. 9. The method of claim 6,wherein each R₁ is —(CH₂)₅CH₃, R₂ is —CH₃, Y is —SO₃ ⁻, X is sodium, andn is
 1. 10. The method of claim 6, wherein each R₁ is —(CH₂)₆CH₃, R₂ is—CH₃, Y is —SO₃ ⁻, X is sodium, and n is
 1. 11. A method of isolating asample, the method comprising: adjusting the sample to pH 6-12; mixing asolvent with a compound of the formula:

wherein R₁ is independently selected from —(CH₂)₀₋₉CH₃, R₂ is selectedfrom the group consisting of —H and —(CH₂)₀₋₅CH₃, Y is selected from thegroup consisting of —SO₃ ⁻ and —PO₃ ⁻, X is a cation is selected fromthe group consisting of sodium, potassium, lithium, and ammonium ions,and n is an integer from 1 to 8; contacting the sample with the mixtureto form a sample-surfactant complex; cleaving the surfactant from thesample-surfactant complex to form cleavage by-products; and isolatingthe sample from the cleavage by-products.
 12. The method of claim 11,wherein the cleaving comprises: adjusting the sample-surfactant complexto pH 2-3; and incubating the sample-surfactant complex for 10-30minutes at 4-50° C.
 13. The method of claim 11, wherein the solvent isselected from the group consisting of water; 0-50% methanol; 0-50%acetonitrile; 5-50 mM ammonium bicarbonate buffer; 5-50 mM Tris-HClbuffer; 5-50 mM sodium phosphate buffer; 5-50 mM ammonium acetatebuffer; and any combination thereof.
 14. The method of claim 11, whereinthe isolating comprises at least one of reversed phase sample clean-uptechniques and solid phase extraction techniques.
 15. The method ofclaim 11 comprising performing electrokinetic transport of thesample-surfactant complex.
 16. The method of claim 15, wherein theperforming electrokinetic transport is electrophoresis.
 17. The methodof claim 11 comprising performing enzymatic digestion of thesample-surfactant complex.
 18. The method of claim 17, wherein theperforming enzymatic digestion comprises: incubating thesample-surfactant complex for less than one hour at 50-60° C.; coolingthe sample-surfactant complex to room temperature; incubating thesample-surfactant complex for less than one hour at less than 30° C. inlimited light; mixing the sample-surfactant complex with an enzyme; andincubating the mixture for less than 24 hours at 20-40° C. with shaking.19. The method of claim 18 comprising desalting the sample-surfactantcomplex with an enzyme.
 20. The method of claim 18 comprising performingmass spectrometry on the isolated sample.